Total concentration of homocysteine in body fluids, such as plasma or serum, is an important marker for disease. For example, homocysteine quantification can be an important risk indicator for cardiovascular disease, can be a sensitive marker of cobalamin and folate deficiencies, and can be used to diagnose in-born errors in metabolism known as homocystinuria. Homocysteine quantification has also been reported as useful in assessing birth defects in pregnant women and cognitive impairment in the elderly. See Frantzen, et al., Enzyme Conversion Immunoassay for Determining Total Homocysteine in Plasma or Serum, Clinical Chemistry 44:2, 311-316 (1998). Current assays, such as those using HPLC or GC-MS, are expensive and require highly skilled technical staff. An efficient and accurate assay, that can be carried out without necessity for highly skilled personnel or complex analytical chemistry equipment, has been needed.
The present invention provides an assay for homocysteine found in plasma, serum or other body fluids of a patient. According to this assay, a homocysteine containing sample is condensed using an enzyme, cystathionine xcex2-synthase, to form cystathionine. This cystathionine is subjected to another enzyme, cystathionine xcex2-lyase, to release pyruvate and ammonia, and homocysteine. The total homocysteine concentration in the patient sample can be determined based on the detection and correlation of the pyruvate and/or ammonia released.
In another embodiment of the invention, the patient sample is subjected to treatment by dithiothreitol or other reducing agent, in appropriate amounts to produce free homocysteine in the sample.
In a still further embodiment, the enzymes cystathionine xcex2-synthase and cystathionine xcex2-lyase may be treated with a phosphorylated form of vitamin B6 in order to optimize their function in the assay of the invention.
A still further embodiment of the present invention is a kit for determining homocysteine concentration in a sample that includes the enzymes cystathionine xcex2-synthase and cystathionine xcex2-lyase, and serine. Such kit may further include a reducing agent such as dithiothreitol (DTT), and an enzyme co-factor such as pyridoxal 5xe2x80x2 phosphate (PLP).